Moderate alcohol preconditioning activates BKCa channels to protect brain damage-induced by cerebral ischemia and reperfusion

OBJCETIVE Epidemiologic studies have demonstrated that consumption of moderate amounts of red wine is associated with significant reductions in incidences of cardiovascular and cerebrovascular diseases,which may be related to alcohol in red wine.Our previous study demonstrated that ethanol ingestion 24 h prior to induction of cerebral ischemic/reperfusion(I/R)reduced delayed neuronal death(DND).Our most recent results supported a role for big Ca2+-sensitive K+channel(BKCa channel)activation in the neuroprotective effects of ethanol preconditioning(Et OH-PC)in global cerebral I/R.Therefore,we hypothesis that moderate Et OH-PC activates BKCa channel to protect brain damage induced by focal cerebral I/R.This project will utilize focal cerebral I/R animal model to explore the function of BKCa channel in Et OH-PC protection in vivo levels by means of pharmacological intervention such as BKCa channel opene(rNS11021,NS)and blocke(rpaxilline,PX).The results will provide theoretical evidence for neuroprotective effect of moderate alcohol preconditioning against ischemic stroke,and the conclusion will also bring to a concept that extrinsic moderate ethanol preconditioning may activate intrinsic protective mechanism in the brain.METHODS The SD rat were randomly divided into the following six groups(n=10):sham,I/R,Et OH-PC+I/R,NS11021-PC+I/R,paxilline+Et OH-PC+I/R,Paxilline+NS11021-PC+I/R.Both Et OH-PC and NS11021-PC(0.1mg·kg-1;ip)were induced 24 h before I/R.The volume of 95%ethanol to be instilled(inμL)was calculated as follows:〔body weight(g)×0.6〕+0.3.This volume of ethanol was mixed in 0.3 m L of sterile distilled water just before administration to the animals by gavage.The Paxilline(2.5 mg·kg-1;ip)was administered 10min beforeEt OH-PC and NS11021-PC.The right middle cerebral artery occlusion(MCAO)was produced by inversion of a 4-0-nylon filament.The filament was withdrawn 2 h after onset of MCAO and then reperfused.Neurological deficits and infarct volume were measured 24 h after I/R.Another 36 rats were randomly divided into 6 groups as above,6 in each group.DWI were performed 2h after ischemic and T2WI MRI were performed 24 h after I/R to observe the infarct volume of brain and the penumbra volume of brain in each group.Then rats were killed and detected the apoptotic cell death and degeneration of neurons.RESULTS Compared to I/R group,the neurological score(P<0.01),the infarct volume of brain(P<0.01),the infarct volume of ischemic penumbra(P<0.01),the percentage of apoptotic cell death(P<0.01)and the percentage of degenerative neurons(P<0.01)were significantly decreased after ethanol preconditioning,while these changes were reversed by paxilline(P<0.05);compared to I/R group,the neurological score(P<0.01),the infarct volume of brain(P<0.01),the infarct volume of ischemic penumbra(P<0.01),the percentage of apoptotic cell death(P<0.01)and the percentage of degenerative neurons(P<0.01)were significantly decreased after NS11021 preconditioning,while these changes were reversed by paxilline(P<0.05).CONCLUSION Our results show that moderate alcohol preconditioning activates BKCa channels to protect brain damage induced by focal cerebral I/R.

Role of BKCa channels in diabetic vascular complications

Objective This review focuses on the role of the large conductance calcium-activated potassium （BKCa） channels in diabetic vascular complications.Data sources Relevant articles published in English or Chinese from 1981 to present were selected from PubMed.The search terms were ＂BKCa channels＂ and ＂diabetes＂.Important references from selected articles were also retrieved.Study selection Articles regarding the role of BKCa channels in diabetic vascular complications and relevant mechanisms were selected.Results The BKCa channels are abundantly expressed in vascular smooth cells and play an important role in regulation of vascular tone.Multiple studies indicated that the expression and function of BKCa channels are altered by different mechanisms in diabetic vascular diseases such as coronary arterial disease,cerebral arterial disease,and diabetic retinopathy.Conclusion BKCa channels may play an important role in diabetic vascular complications and may be an effective therapeutic target for relieving and reducing the burden of diabetic vascular complications.

Glabridin Relaxes Vascular Smooth Muscles by Activating BK_(Ca) Channels and Inhibiting Phosphodiesterase in Human Saphenous Vein

The aim of the current study was to investigate the pharmacological activity of glabridinon the isolated human saphenous vein (SV) and explore the underlying mechanisms. Samples of patients' SVs were removed during bypass surgery, and 4-mm lengths of the vessels were placedin Krebs solution at ±4℃ and hung in an isolated organ bath to assess their contraction/relaxationresponses. The contraction/relaxation responses were recorded to observe if the cyclic guanosinemonophosphate (cGMP)/protein kinase G (PKG) pathway mediates the relaxant effect of glabridinafter treatment with blockers like ODQ (a guanylate cyclase inhibitor), KT5823 (a PKG inhibitor),isobutylmethylxanthine [IBMX, a phosphodiesterase (PDE) inhibitor], and cantharidin [Cant,a myosin light-chain phosphatase (MLCP) inhibitor]. Moreover, nitric oxide (NO), cGMP, andPKG levels in SV tissues were determined by ELISA after incubation with glabridin, N(o)-nitro-L-arginine methyl ester (L-Name, a NO synthetase inhibitor), phenylephrine (PE), ODQ, IBMX,and KT5823. The results showed that glabridin relaxed the vascular smooth muscle of humanSV pretreated with PE in a dose-dependent manner, which was independent of the endothelium.The vasorelaxant effect of glabridin was only inhibited by iberiotoxin (IbTX), Cant, and KT5823.Glabridin increased cGMP and PKG levels in SV homogenates, whereas it did not alter the NOlevel. The enhancing efects of cGMP and PKG levels by glabridin were abolished by ODQ andKT5823. In conclusion, glabridin has a vasorelaxant effect, which is associated with the activationof BKc. channels and inhibition of PDE.

Low-Dose Ethanol Preconditioning Protects Against Oxygen-Glucose Deprivation/Reoxygenation-Induced Neuronal Injury By Activating Large Conductance,Ca2+-Activated K+Channels In Vitro

Increasing evidence suggests that low to mod- erate ethanol ingestion protects against the deleterious effects of subsequent ischemia/reperfusion; however, the underlying mechanism has not been elucidated. In the present study, we showed that expression of the neuronal large-conductance, Ca2＋-activated K＋ channel （BKca） α- subunit was upregulated in cultured neurons exposed to oxygen-glucose deprivation/reoxygenation （OGD/R） compared with controls. Preconditioning with low-dose ethanol （10 mmol/L） increased cell survival rate in neurons subjected to OGD/R, attenuated the OGD/R-induced elevation of cytosolic Ca2＋ levels, and reduced the number of apoptotic neurons. Western blots revealed that ethanol preconditioning upregulated expression of the anti-apoptotic protein Bcl-2 and downregulated the pro-apoptotic protein Bax. The protective effect of ethanol precondi- tioning was antagonized by a BKα channel inhibitor, paxilline. Inside-out patches in primary neurons also demonstrated the direct activation of the BKCa channel by 10 mmol/L ethanol. The above results indicated that low- dose ethanol preconditioning exerts its neuroprotective effects by attenuating the elevation of cytosolic Ca2＋ and preventing neuronal apoptosis, and this is mediated by BKca channel activation.

Irrelevance of BK_(Ca) Channel Expression to VSMCs Phenotype under Shear Stress

Large conductance Ca^(2+)-activated K^+ (BK_(Ca)) channel exhibits a phenotype-dependent expression on vascular smooth muscle cells(VSMCs), which prefers to contractile phenotype. Meanwhile, shear stress definitely influences VSMCs proliferation and contraction. Thereby, a hypothesis was raised, would shear stress change the BK_(Ca)expression and correlate with VSMC phenotype? In order to investigate it, VSMCs were exposed to shear stress in a parallel-plate flow chamber with 12 dynes/cm^2 for 12 h. Subsequently, the effect of shear stress on VSMC proliferation, BK_(Ca)channel expression and contractile phenotype marker, α-smooth muscle cell actin(α-SMA) and smooth muscle myosin heavy chain(SMMHC), was determined by immunofluorescence microscopy, flow cytometery as well as reverse transcriptionpolymerase chain reaction(RT-PCR), respectively. Data show that shear stress enhanced the expression of BK_(Ca)channel while inhibiting VSMC proliferation.Paralleled to those phenomena, the expression of both α-SMA and SM-MHC were decreased significantly. These results demonstrated that upregulation of BK_(Ca)channel was irrelevant to the maintenance VSMC of contractile phenotype under shear stress.This finding provides a new insight into understanding the correlation of BK_(Ca)channel and VSMC phenotype.